Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester

ABSTRACT

Disclosed is a method for the preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl esters, comprising the enantioselective enzyme reduction of a 1-aryl-2-tetrazolyl-ethyl ketone to form a (R)-1-aryl-2-tetrazolyl-ethyl alcohol and the carbamation of said alcohol.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/904,267, filed Feb. 14, 2010, which claims priority for U.S.Provisional Application No. 61/251,867, filed Oct. 15, 2009, thedisclosure of which is incorporated herein in its entirety.

The present invention relates to a method for the preparation ofcarbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester. More particularly,the present invention relates to a method for preparing carbamic acid(R)-1-aryl-2-tetrazolyl-ethyl ester, comprising the enantioselectiveenzyme reduction of an arylketone.

BACKGROUND OF THE INVENTION

As disclosed in U.S. Patent Application Publication No. 2006/0258718 A1,carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl esters (hereinafter referredto as “the carbamate compounds”) possess anticonvulsant activity and areuseful in the treatment of disorders of the central nervous system,especially including anxiety, depression, convulsion, epilepsy,migraines, bipolar disorder, drug abuse, smoking, ADHD, obesity, sleepdisorders, neuropathic pain, strokes, cognitive impairment,neurodegeneration, strokes and muscle spasms.

Depending on the position of N in the tetrazole moiety thereof, thecarbamate compounds are divided into two positional isomers:tetrazole-1-yl (hereinafter referred to as “1N tetrazole”) andtreatzole-2-yl (hereinafter referred to as “2N tetrazole”). Theintroduction of tetrazole for the preparation of the carbamate compoundsresults in a 1:1 mixture of the two positional isomers which arerequired to be individually isolated for pharmaceutical use.

Having chirality, the carbamate compounds must be in high optical purityas well as chemical purity as they are used as medications. In thisregard, U.S. Patent Application Publication No. 2006/0258718 A1 uses thepure enantiomer (R)-aryl-oxirane as a starting material, which isconverted into an alcohol intermediate through a ring-opening reactionby tetrazole in the presence of a suitable base in a solvent, followedby introducing a carbamoyl group into the resulting alcoholintermediate. For isolation and purification of the 1N and 2N positionalisomers thus produced, column chromatography is utilized after theformation of an alcohol intermediate or carbamate.

For use in the preparation described above, (R)-2-aryl-oxirane may besynthesized from an optically active material, such as substituted(R)-mandelic acid derivative via various route, or obtained byasymmetric reduction-ring formation reaction of α-halo arylketone, or byseparation of racemic 2-aryl-oxirane mixture into its individualenantiomers. As such, (R)-2-aryl-oxirane is an expensive compound.

In addition, the ring-opening reaction of (R)-2-aryl-oxirane withtetrazole is performed at relatively high temperatures because of thelow nucleophilicity of the tetrazole. However, because tetrazoles startto spontaneously degrade at 110˜120° C., the ring opening reactionincludes the highly likely risk of a runaway reaction.

In terms of a selection of reaction, as there are two reaction sites ineach (R)-2-aryl-oxirane and tetrazole, the ring-opening reactiontherebetween affords the substitution of 1N- or 2N-tetrazole at thebenzyl or terminal position, resulting in a mixture of a total of 4positional isomers. Therefore, individual positional isomers are low inproduction yield and difficult to isolate and purify.

SUMMARY OF THE INVENTION

In accordance with the present invention, the foregoing disadvantages ofthe prior art are overcome by a novel method for preparing carbamic(R)-1-aryl-2-tetrazolyl-ethyl esters. In the present method, a(R)-1-aryl-2-tetrazolyl-ethyl alcohol represented by Chemical Formula 3is formed by the enantioselective enzymatic reduction of an arylketone,represented by Chemical Formula 2, and the alcohol is then carbamated toform the carbamic acid (R)-1-aryl-2-tetrazolyl ethyl ester, representedby Chemical Formula 1:

wherein, R₁ and R₂ are independently selected from a group consisting ofhydrogen, halogen, perfluoroalkyl, alkyl of 1 to 8 carbon atoms,thioalkoxy of 1 to 8 carbon atoms, and alkoxy of 1 to 8 carbon atoms;and one of A₁ and A₂ is CH with the other being N.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with an embodiment of the present invention, a methodcomprising enantioselective enzymatic reduction of an arylketonerepresented by the following Chemical Formula 2 and the carbamation ofthe resultant alcohol compound represented by the following ChemicalFormula 3 is provided for the preparation of carbamic acid(R)-1-aryl-2-tetrazolyl-ethyl ester, represented by the followingChemical Formula 1.

wherein,

R₁ and R₂ are independently selected from a group consisting ofhydrogen, halogen, perfluoroalkyl, alkyl of 1 to 8 carbon atoms,thioalkoxy of 1 to 8 carbon atoms, and alkoxy of 1 to 8 carbon atoms;and

one of A₁ and A₂ is CH with the other being N.

The arylketone of Chemical Formula 2, useful as a starting material inthe preparation method of the present invention, may be synthesized by,for example, a substitution reaction between the arylketone of ChemicalFormula 4 and tetrazole of Chemical Formula 5:

wherein,

R₁ and R₂ are as defined above; and

X is a leaving group, such as a halide or sulfonate.

An economical advantage is given to the synthesis of the arylketones ofChemical Formula 2 from the compounds represented by Chemical Formulae 4and 5 because they are commercially available, relatively inexpensivecompounds. In addition, the substitution reaction can be carried out inrelatively mild conditions, compared to the ring-opening reactionbetween (R)-2-aryl-oxirane and tetrazole. The method according to thepresent invention is therefore certain of process safety, althoughemploying potentially explosive tetrazole, and ensures high productionyield and easy purification, with the production of no unnecessarypositional isomers at benzyl positions.

The arylketone represented by Chemical Formula 2 which can besynthesized by the substitution reaction with tetrazole may be in amixture of positional isomers including 1N arylketone of the followingChemical Formula 2a and 2N arylketone of the following Chemical Formula2b, which can be isolated and purified through commercially availablecrystallization.

The crystallization useful in the present invention may comprise addinga solubilizing agent to the product of the substitution reaction, thatis a mixture of the positional isomers, and then adding a precipitatingagent. Optionally, the crystallization may further comprise, after theprecipitation, filtrating the precipitate, concentrating the filtrateand adding an additional precipitating agent.

Illustrative, non-limiting examples of the solubilizing agent includeacetone, acetonitrile, tetrahydrofuran, ethyl acetate, dichloromethane,chloroform, 1,4-dioxane, and lower alcohols of 1 to 4 carbon atoms, andcombinations thereof. The solubilizing agent may be used in an amount offrom 0 to 20 ml (v/w) based on the weight (g) of the mixture of thepositional isomers. As used herein, the addition of the solubilizingagent in an amount of zero ml (v/w) is intended to mean immediatelyadding the subsequent precipitating agent without dilution of thefiltrate.

Examples of the precipitating agent include water, C1-C4 lower alcohol,diethylether, pentane, hexane, cyclohexane, heptane and combinationsthereof, but are not limited thereto. The precipitating agent may beslowly added in an amount of from zero to 40 ml (v/w) based on theweight (g) of the mixture of positional isomers. As used herein, theaddition of the precipitating agent in an amount of zero ml is intendedto mean allowing the reaction mixture to stand, or cooling it withoutthe addition of the precipitating agent to yield the precipitates.Filtration of the precipitates yields the 1N arylketone of ChemicalFormula 2a as crystals with high purity.

On the other hand, the filtrate thus obtained after the filtration stepmay be concentrated to increase the ratio of the precipitating agent tothe solubilizing agent, thereby yielding the 2N arylketone of ChemicalFormula 2b with high purity. The concentration ratio of the filtrate canbe suitably determined by those of ordinary skill in the art. Forexample, concentration is carried until the solvent is totally removedoff, then the solubilizing agent and the precipitating agent are addedas described above. Unlike column chromatography, this crystallizationmay be commercially used without much difficulty.

The enantioselective enzymatic reduction according to the presentinvention allows for the conversion of the arylketone of ChemicalFormula 2 above into the alcohol compound with (R)-configuration,represented by the Chemical Formula 3 above. The enantioselectiveenzymatic reduction may be performed using an oxidoreductase enzyme thatis in suspension in the reaction mixture, or immobilized in aconventional manner. The enzyme may be utilized in a completely purifiedstate, in a partially purified state, or in the microbial cells is whichit was expressed. The cells themselves may be in a native state, apermeabilized state or a lysed state. It will be appreciated by those ofordinary skill in the art that use of the enzyme in the cells ispreferred for the practice of the process of the invention since itrepresents a significant savings in cost. Most preferably, the enzyme isexpressed in E. coli and used as a suspension of native cells.

The process of enzymatic reduction of the aryl ketone compounds ofFormula 2 can be performed in a reaction mixture comprising saidcompound of Chemical Formula 2, an oxidoreductase, NADH or NADPH as acofactor, a cosubstrate and a suitable buffer wherein the oxidoreductasecomprises an amino acid sequence wherein at least 60% of the amino acidsare identical with one of the amino acid sequences SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3 or SEQ ID NO:4.

It has been found that polypeptides comprising one of amino acidsequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4 or apolypeptides comprising an amino sequence which is identical by at least60%, preferably at least 90% to one of the amino acid sequences SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4 and possessingoxidoreductase activity can be used for reducing the compound of Formula2 to the compound of Formula 3 (R-configuration) with high conversionand high enantiomeric selectivity. The enantiomeric excess of theR-alcohol formed in the enantioselective enzymatic reduction is at leastabout 89%, preferably at least about 95% and most preferably at leastabout 99%.

The organism producing the oxidoreductase polypeptides useful in theenantioselective enzymatic reduction may be a wild strain or a variantand is preferably selected from Candida magnolia, Candida vaccinii, andOryctolagus cuniculus. Yeast of the Candida genus is preferred forproducing the oxidoreductase enzymes utilized in the present process.Derivatives of the polypeptides are those having at least sixty percenthomology with the SEQ IDs given above and possessing oxidoreductaseactivity. Those skilled in the art are aware that there are systems andtechnology available to accurately determine sequence homology.

A polypeptide comprising SEQ ID NO: 1 may be encoded by a DNA sequenceSEQ ID NO:5 which is obtainable, for example, from the organismOryctolagus cuniculus deposited under the conditions of the BudapestTreaty with the Deutsche Sammlung für Mikroorganismen und Zellkulturen,Mascheroder Weg 1b, 38124 under the number DSMZ 22167, specifically fromrabbit DSMZ 22167, or by a nucleic acid sequence that hybridizestherewith. A polypeptide comprising SEQ ID NO:2 may be encoded by a DNAsequence SEQ ID NO:6 which is obtainable, for example, from the organismCandida magnoliae DSMZ 22052, or by a nucleic acid sequence thathybridizes therewith.

A polypeptide comprising SEQ ID NO:3 may be encoded by a DNA sequenceSEQ ID NO:7, which is obtainable, for example, from the organism Candidavaccinii CBS7318, or by a nucleic acid sequence that hybridizestherewith. A polypeptide comprising SEQ ID NO:4 may be encoded by a DNAsequence SEQ ID NO: 8, which is obtainable, for example, from theorganism Candida magnoliae CBS6396, or by a nucleic acid sequence thathybridizes therewith.

The oxidoreductase having one of polypeptide sequences mentioned aboveis obtained in useable quantities by conventional procedures recognizedby those skilled in the art. A polynucleotide coding for the amino acidsequence is cloned into a suitable vector and thereafter introduced intoa host organism capable of expressing the gene coding for the sequence.Microorganisms susceptible of transforming to become capable ofexpressing of such a peptide are well known in the art. A preferredmicroorganism is Escherichia coli. As stated above, the oxidoreductaseexpressed by transformed E. coli may be extracted from the E. coli cellsand partially or completely purified for use in the process, or may beutilized in the cells themselves which may be in a native, permeabilizedor lysed state. A preferred embodiment of the enantioselective enzymaticreduction of the present invention utilizes a suspension of theoxidoreductase as cells in the native state. Any of these forms may beutilized in the free or immobilized form.

The reduction reaction may be carried out in a single phase systemhaving the cells containing the enzyme suspended therein. Alternatively,the reaction may be performed in a two-phase aqueous/organic solventsystem as described in U.S. Patent Application Publication No.2009/0017510 and U.S. Pat. No. 7,371,903. The reaction may be carriedout as a conventional batch reaction, or as a continuous process. Itwill be appreciated that one of the significant advantages of theenantioselective enzymatic reduction for commercial applications is thatit is amenable to continuous operation.

The reaction mixture preferably contains from about 35 g to 350 g ofcells per kg of reactant added therein. The suspension is the aqueousportion of the reaction mixture which also contains a buffer, forexample a TEA (triethanolamine), phosphate, Tris/HCl or glycine buffer.The buffer may additionally comprise ions for the stabilization of theenzyme, for example, a source of magnesium ions. Additional additivesthat may be present in the buffer for stabilizing the enzymes mayinclude a polyol, such as glycerol, sorbitols and the like, sulfurcompounds, such as 1,4-DL-dithiothreitol, glutathione, cysteine or thelike, amino acids and peptides, or detergents, such as DMSO. A preferredstabilizer for the enzyme is a polyol, particularly glycerol, which maybe present in from about 10% to 80% by weight, preferably about 50% byweight, based on the weight of the cell suspension.

The enantioselective enzymatic reduction process is advantageouslycarried out using a coupled substrate principle wherein the reactionmixture utilizes a second substrate for the regeneration of thecofactor, or coenzyme, which functions to provide hydrogen for thereduction of the arylketone substrate. The cofactor is preferablynicotineamide adenine dinucleotide phosphate (NADP) or nicotineamideadenine dinucleotide (NAD), which are utilized in the reduced state,i.e. NADPH or NADH, respectively. The cofactor is present in thereaction mixture in a concentration of from about 0.01 mM to 5 mM,preferably 0.05 mM to 0.5 mM. In the reaction, the second substratefunctions by being oxidized in the regeneration of the NADPH or NADHcofactor. The second substrate is a secondary alcohol represented by theFormula R_(x)R_(y)CHOH, wherein R_(x) represents carbon with x being aninteger from 1-10 and R_(y) represents hydrogen with y being an integerequal to two times the value of x plus two. Examples of suitable secondsubstrate include 2-propanol, 2-butanol 4-methyl-2-pentanol, 2-pentanol,2-heptanol, 2-octanol and the like. A preferred second substrate is2-butanol. The second substrate is present in the reaction mixture infrom about 10% to 80% by volume, preferably from about 40% to 60% byvolume, most preferably about 50% by volume.

The oxidized cofactor formed during the reduction of the arylketone isregenerated by oxidation of the second substrate, which also can becatalyzed by the oxidoreductase. Thus, a particular economic advantageof the present process is that the oxidoreductase affects both reductionof the arylketone of Formula 1 and oxidation of the second substrate,therefore no further enzyme has to be used for cofactor regeneration. Itis also within the scope of the present invention to add another enzymeto the reaction mixture for cofactor regeneration in order to enhancethe rate of reduction of the aryl ketone.

In a further embodiment, an organic solvent that is not involved in theregeneration of the cofactor may be added to the reaction mixture andthe reduction process carried out in an aqueous organic 2-phase system.Examples of such solvents include, without intended limitation, diethylether, tertiary butyl methyl ether, diisopropyl ether, dibutyl ether,ethyl acetate, butyl acetate, heptane, hexane or cyclohexane. Such asolvent may be present in from about 1% to 50% by volume based on thevolume of the reaction mixture.

The amount of the arylketone substrate in the reaction mixture ispreferably greater than about 0.1% by weight and may be increased toabout 50% by weight, with a preferred concentration being from about 5to 30% by weight. The amount of the substrate will vary depending on thepurity thereof since the process may be carried out with the substratein a purified state or as raw product containing varying amounts andtypes of impurities. The pH of the reaction mixture after the additionof all components will be in the range of 5 to 10, preferably from 7 to9, and optimally about pH 8. The enzymatic reduction according to thepresent invention is carried out at a temperature of from about 10-45°C., preferably from about 20-40° C., most preferably from about 25-35°C.

The enantioselective reduction process is cost-effective andenvironment-friendly in addition to providing the alcohols of Formula 3in high yield and very high enantioselectivity. Thus, an alcoholcompound with an (R)-configuration of high optical purity can beobtained in the presence of the enzyme under the above-mentionedreaction conditions within from about 12 to 96 hours, preferably fromabout 24 to 48 hours. During the incubation, the pH of the mixture ismaintained within the ranges given above by periodic testing and theaddition of a conventional acidic or basic reagents, for example sodiumcarbonate and sodium hydroxide, respectively. The efficiency of theenantioselective enzymatic reduction can be expressed by the totalturnover number (TTN) which is the moles of the chiral alcohol ofFormula 2 produced per mole of cofactor used. The TTN of theenantioselective enzymatic reduction is from about 10² to 10⁵,preferably ≧10³.

When the alcohol compound obtained through the enantioselectiveenzymatic reduction exists as a positional isomer mixture of 1N alcoholof Chemical Formula 3a and 2N alcohol of Chemical Formula 3b, it can beisolated and purified into individual positional isomers of high purityby crystallization:

The crystallization may comprise adding a solubilizing agent to thepositional isomer mixture resulting from the reduction; and adding aprecipitating agent, and optionally filtering the precipitate; andconcentrating the filtrate and adding an additional precipitating agent.

Examples of the solubilizing agent useful in the crystallization includeacetone, acetonitrile, tetrahydrofuran, ethyl acetate, dichloromethane,chloroform, 1,4-dioxane, lower alcohol of 1 to 4 carbon atoms, andmixtures thereof, but are not limited thereto. The solubilizing agentmay be added in an amount of zero to 20 ml (v/w) based on the weight (g)of the positional isomer mixture.

Non-limiting examples of the precipitating agent include water, a loweralcohol of 1 to 4 carbon atoms, diethylether, pentane, hexane,cyclohexane, heptane, and mixtures thereof. The precipitating agent maybe slowly added in an amount of from zero to 40 ml (v/w) based on theweight (g) of the positional isomer mixture.

Following the addition of the precipitating agent, filtration may yield1N alcohol (3a) as a precipitate of high purity.

Furthermore, 2N alcohol (3b) can be obtained as a crystal form of veryhigh purity by concentrating the filtrate and increasing the ratio ofthe precipitating agent to the solubilizing agent.

These crystallization steps may be omitted when the positional isomersof arylketone of Chemical Formula 2 are already isolated and purified.

The introduction of a carbomoyl moiety into the alcohol compound with(R)-configuration of Chemical Formula 3 leads to carbamate with(R)-configuration, represented by Chemical Formula 1:

wherein,

R₁ and R₂ are independently selected from a group consisting ofhydrogen, halogen, perfluoroalkyl, an alkyl of 1 to 8 carbon atoms, athioalkoxy of 1 to 8 carbon atoms, and an alkoxy of 1 to 8 carbon atoms;and one of A₁ and A₂ is CH with the other being N.

In the carbamation step, for example, inorganic cyanate-organic acid,isocyanate-water, or carbonyl compound-ammonia may be employed tointroduce a carbamoyl moiety.

For the carbamation with inorganic cyanate-organic acid, the alcoholcompound with (R)-configuration of Chemical Formula 3 is dissolved in anorganic solvent, for example, diethylether, tetrahydrofuran,1,4-dioxane, acetonitrile, dichloromethane, chloroform or mixturesthereof, and mixed with 1 to 4 equivalents of inorganic cyanate such assodium cyanate and an organic acid, such as methane sulfonic acid oracetic acid, followed by reacting at about −10 to 70° C.

With regard to use of the isocyanate-water, 1 to 4 equivalents ofisocyanate, for example, chlorosulfonic isocyanate, trichloroacetylisocyanate, trimethylsilyl isocyanate, are added to a solution of thealcohol compound with (R)-configuration of Chemical Formula 3 in anorganic solvent, for example, diethylether, tetrahydrofuran,1,4-dioxane, acetonitrile, dichloromethane, chloroform or mixturesthereof, and reacted at about −50 to 40° C. Subsequently, withoutpurification, 1 to 20 equivalents of water are added to inducehydrolysis.

With regard to use of the carbonyl compound-ammonia, 1 to 4 equivalentsof a carbonyl compound, for example, 1,1′-carbonyldiimidazole, carbamolychloride, N,N-disuccinimidyl carbonate, phosgene, triphosgene, orchloroformate, are added to a solution of the alcohol compound with(R)-configuration of Chemical Formula 3 in an organic solvent, forexample, diethylether, tetrahydrofuran, 1,4-dioxane, acetonitrile,dichloromethane, chloroform or mixtures thereof, and reacted at about−10 to 70° C., followed by adding 1 to 10 equivalents of ammonia withoutpurification.

After the carbamation, the carbamate compound of Chemical Formula 1 thusobtained may be purified to higher optical and chemical purity throughthe following crystallization. The crystallization comprises adding asolubilizing agent to the product of the carbamation; and then adding aprecipitating agent, and optionally filtering the precipitate and addingan additional precipitating agent. For pharmaceutical use, it ispreferable that there is always a final purification of the carbamatedproduct before use, but that there can be a crystallization step earlierin the process.

Non-limiting examples of the solubilizing agent include acetone,acetonitrile, tetrahydrofuran, ethyl acetate, dichloromethane,chloroform, 1,4-dioxane, lower alcohol of 1 to 4 carbon atoms, andmixtures thereof. Based on the weight (g) of the reaction product, thesolubilizing agent may be used in an amount of from zero to 20 ml (v/w).

Non-limiting examples of the precipitating agent include water, loweralcohols of 1 to 4 carbon atoms, diethylether, pentane, hexane,cyclohexane, heptane and mixtures thereof. Based on the weight (g) ofthe reaction product, the precipitating agent may be slowly added in anamount of from zero to 40 ml (v/w).

Comprising enantioselective enzymatic reduction, the method of thepresent invention can provide optically high pure carbamate compounds.In addition, the mild reaction conditions which the method of thepresent invention requires ensure process safety. Furthermore, thecrystallization step applicable to large-scale production before orafter the enantioselective enzymatic reduction or after the carbamationresults in a higher chemical purity of the carbamate compounds. Thecarbamate compounds prepared according to the present invention are veryuseful in the treatment of CNS disorders such as convulsion.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as in any way limiting the present invention.

Preparation Example 1 Preparation of1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-1-yl)ethan-1-one

To a suspension of 2-bromo-2′-chloroacetophenone (228.3 g, 0.978 mol)and potassium carbonate (161.6 g, 1.170 mol) in acetonitrile (2000 mL)was added a 35 w/w % 1H-tetrazole dimethylformamide solution (215.1 g,1.080 mol) at room temperature. These reactants were stirred for 2 h at45° C. and distilled under reduced pressure to remove about 1500 mL ofthe solvent. The concentrate was diluted in ethyl acetate (2000 mL) andwashed with 10% brine (3×2000 mL). The organic layer thus separated wasdistilled under reduced pressure to afford 216.4 g of an oily solidresidue. To a solution of the solid residue in ethyl acetate (432 mL)was slowly added heptane (600 mL). The precipitate thus formed wasfiltered at room temperature and washed to yield 90.1 g (0.405 mol) of1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-1-yl)ethan-1-one (hereinafterreferred to as “1N ketone”).

¹H-NMR (CDCl₃) δ8.87 (s, 1H), d 7.77 (d, 1H), d 7.39-7.62 (m, 3H), d5.98 (s, 2H)

Preparation Example 2 Preparation of1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-one

After the filtration of Preparation Example 1, the filtrate wasconcentrated and dissolved in isopropanol (100 mL), and to which heptane(400 mL) was then added to complete the crystallization. Filtering andwashing at 5° C. afforded1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-one (hereinafterreferred to as “2N ketone”) as a solid. 94.7 g (0.425 mol).

¹H-NMR (CDCl₃) d 8.62 (s, 1H), d 7.72 (d, 1H), d 7.35-7.55 (m, 3H), d6.17 (s, 2H)

Preparation Example 3 Preparation of Alcohol Compound of(R)-Configuration by Enantioselective Enzymatic Reduction Via VariousOxidoreductases

The following four solutions were prepared as follows:

Enzyme Solution 1

Competent Escherichia coli StarBL21(De3) cells (Invitrogen) weretransformed with the expression constructs pET21-MIX coding foroxidoreductase SEQ ID NO 1. The Escherichia coli colonies transformedwith the resulting expression constructs were then cultivated in 200 mLof LB medium (1% tryptone, 0.5% yeast and 1% sodium chloride) with 50micrograms/mL of ampicillin or 40 micrograms/mL of kanamycin,respectively, until an optical density of 0.5, measured at 550 nm, wasachieved. The expression of the desired recombinant protein was inducedby the addition of isopropylthiogalactoside (IPTG) to a concentration of0.1 mM. After 16 hours of induction at 25° C. and 220 rpm, the cellswere harvested and frozen at −20° C. In the preparation of the enzymesolutions, 30 g of cells were resuspended in 150 mL of triethanolaminebuffer (TEA 100 nM, 2 mM MgCl₂, 10% glycerol, pH 8) and homogenized in ahigh pressure homogenizer. The resultant enzyme solution was mixed with150 mL glycerol and stored at −20° C.

Enzyme Solution 2

RB791 cells (E. coli genetic stock, Yale, USA) were transformed with theexpression constructs pET21-MLX coding for oxidoreductase SEQ ID NO 2.The Escherichia coli colonies transformed with the resulting expressionconstructs were then cultivated in 200 mL of LB medium (1% tryptone,0.5% yeast and 1% sodium chloride) with 50 micrograms/mL of ampicillinor 40 micrograms/mL of kanamycin, respectively, until an optical densityof 0.5, measured at 550 nm, was achieved. The expression of the desiredrecombinant protein was induced by the addition ofisopropylthiogalactoside (IPTG) to a concentration of 0.1 mM. After 16hours of induction at 25° C. and 220 rpm, the cells were harvested andfrozen at −20° C. In the preparation of the enzyme solutions, 30 g ofcells were resuspended in 150 mL of triethanolamine buffer (TEA 100 nM,2 mM MgCl₂, 10% glycerol, pH 8) and homogenized in a high pressurehomogenizer. The resultant enzyme solution was mixed with 150 mLglycerol and stored at −20° C.

Enzyme Solution 3

Enzyme solution 3 was prepared in the same manner as described in Enzymesolution 1 except that expression constructs pET21-MIX coding foroxidoreductase SEQ ID NO 3 instead of expression constructs pET21-MLXcoding for oxidoreductase SEQ ID NO 1 was used.

Enzyme Solution 4

Enzyme solution 4 was prepared in the same manner as described forenzyme solution 2 except that expression constructs pET21-MLX coding foroxidoreductase SEQ ID NO 4 instead of expression constructs pET21-MLXcoding for oxidoreductase SEQ ID NO 2 was used.

Different oxidoreductases contained in each of enzyme solutions 1 to 4were examined as follows for the conversion of1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-1-yl)ethan-1-one (1N ketone) and1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-one (2N ketone) tothe corresponding 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-1-yl)ethan-1-ol(hereinafter, referred to as “1N alcohol”) and1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-ol (hereinafter,referred to as “2N alcohol”), respectively.

Reaction Batch A

160 μl buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8) 100 μl NADPH(40 mg/ml) 40 μl 2-propanol 50 μl enzyme solution 1 2 mg 1N ketone or 2NketoneReaction Batch B

160 μl buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8) 100 μl NADPH(40 mg/ml) 40 μl 2-propanol 50 μl enzyme solution 2 2 mg 1N ketone or 2NketoneReaction Batch C

350 μl buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8) 0.05 mg NADP50 μl enzyme solution 3 10 mg 1N ketone or 2N ketone 250 μl4-methyl-2-pentanol 50 μl enzyme (oxidoreductase from Thermoanerobiumbrockii) solution for regeneration of cofactorReaction Batch D

350 μl buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8) 0.05 mg NADP50 μl enzyme solution 4 10 mg 1N ketone or 2N ketone 250 μl4-methyl-2-pentanol 50 μl enzyme (oxidoreductase from Thermoanerobiumbrockii) solution for regeneration of cofactor

After 24 h of incubating each reaction batch A, B, C and D, 1 mL ofacetonitrile was added to each reaction batch which was centrifuged andtransferred into a HPLC analysis vessel for enantiomeric excess andconversion. Conversion and ee-value of products are listed in Table 1below calculated using the following equations:Conversion Rate (%)=[(Area of Product)/(Area of Reactant+Area ofProduct)]×100ee-value(%)=[(Area of R-Configuration−Area of S-Configuration)/(Area ofR-Configuration+Area of S-Configuration)]×100

TABLE 1 ee-values % ee(enantiomer) Conversion R-2N R-1N Reaction batchused (% of reduced ketone) Alcohol, 2b Alcohol, 2a Reaction batch A >9889(R) >99(R) Reaction batch B >98 >99(R)   >99(R) Reaction batch C >9895(R) >99(R) Reaction batch D >98 98(R)   95(R)

Preparation Example 4 Enzymatic Reduction Via Oxidoreductase SEQ NO: 2

For the conversion of 1N/2N ketone to R-1N/R-2N alcohol, 30 μl of theenzyme solution 2 containing the oxidoreductase SEQ NO: 2 were added toa mixture of 300 μl of a buffer (100 mM TEA, pH 8, 1 mM MgCl₂, 10%glycerol), 100 mg of a mixture of 1N ketone and 2N ketone(1N:2N=14%:86%), 0.04 mg NADP and 300 μl 2-butanol. The reaction mixturewas incubated at room temperature under constant thorough mixing. After48 hours, more than 98% of the ketones were reduced to an alcoholmixture of the following composition (R-2N alcohol 80%; S-2N alcohol 0%;R-1N alcohol 20%, S-1N alcohol 0%; 1N ketone 0%; 2N ketone 0%).

After general work up and recrystallization with ethyl acetate/hexane,optically pure alcohols were obtained as below:

-   (R)-1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-1-yl)ethan-1-ol (1N    alcohol)

¹H-NMR (CDCl₃) d 8.74 (s, 1H), d 7.21-7.63 (m, 4H), d 5.57 (m, 1H), d4.90 (d, 1H), d 4.50 (d, 1H), d 3.18 (d, 1H);

-   (R)-1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-ol (2N    alcohol)

¹H-NMR (CDCl₃) d 8.55 (s, 1H), d 7.28-7.66 (m, 4H), d 5.73 (d, 1H), d4.98 (d, 1H), d 4.83 (d, 1H), d 3.38 (br, 1H).

Preparation of Carbamate Preparation Example 5 Preparation of CarbamicAcid (R)-1-(2-Chlorophenyl)-2-(tetrazol-2-yl)ethyl ester

50 ml of the enzyme solution 2 containing the oxidoreductase SEQ NO: 2were added to a mixture of 250 ml of a buffer (100 mM TEA, pH 8, 1 mMMgCl2, 10% glycerol), 50 g (225 mmol) of1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-one(2N ketone), 4 mgNAD, 300 ml of 2-propanol and 150 mL of butyl acetate. The reactionmixture was stirred at room temperature. After 48 hours more than 98% of2N ketone was reduced to corresponding(R)-1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-ol (R-2Nalcohol) with >99% ee values. To this resulting mixture, 500 mL of ethylacetate was added. After being separated, the organic layer thus formedwas washed with 10% brine (3×500 mL). The organic layer thus formed wasdried over magnesium sulfate and filtered and the filtrate was distilledunder reduced pressure to give 50.4 g (224 mmol) of1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-ol (R-2N alcohol,optical purity 99.9%) as an oily residue. To this resulting crudeproduct, 450 mL of tetrahydrofuran was added. After cooling to −15° C.,38 g (267 mmol) of chlorosulfonyl isocyanate was slowly added andstirred at −10° C. for 2 h. The slow addition of water inducedtermination of the reaction. The resulting solution was concentratedunder reduced pressure until about 300 mL of the solvent was removed.The concentrate was diluted with 600 mL of ethyl acetate and washed with10% brine (3×500 mL). The organic layer was concentrated under reducedpressure and the concentrate was dissolved in isopropanol (90 mL) towhich heptane (180 mL) was slowly added, leading to the completion ofcrystallization. The precipitate thus obtained was filtered and washedto afford 51.8 g (194 mmol) of carbamic acid(R)-1-(2-chlorophenyl)-2-(tetrazol-2-yl)ethyl ester (optical purity99.9%).

¹H-NMR (Acetone-d₆) d 8.74 (s, 1H), d 7.38-7.54 (m, 4H), d 6.59 (m, 1H),d 6.16 (Br, 2H), d 4.90 (d, 1H), d 5.09 (m, 2H)

As described hitherto, carbamate compounds with high optical andchemical purity can be produced with an economical benefit in accordancewith the present invention.

Although the preferred embodiments of the present invention have beendisclosed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

AMINO ACID SEQUENCESSEQ ID NO 1: Oryctolagus cuniculus from rabbit DSMZ 22167 1massgvtrrd plankvaivt astdgiglai arrlaqdgah vvissrkqqn vdravaalqa 61eglsvtgtvc hvgkaedrer lvatalnlhg gidilvsnaa vnpffgklmd vteevwdkil 121dinvkamalm tkavvpemek rgggsvviva siaafnpfsg lgpynvskta lvgltknlal 181elaaqnirvn clapglikts fskalwedka qeeniiqklr irrlgkpeec agivsflcse 241dasyitgetv vvaggapsrlSEQ ID NO 2: Candida magnoliae DSMZ 22052 protein sequence carbonyl reductase1 msatsnalit gasrgmgeat aiklalegys vtlasrgieq lnaikeklpi vkkgqqhyvw 61qldlsdieaa stfkqaplpa ssydvffsna gvvdfapfad qsetaqkdlf tvnllspval 121tktivkaiad kpretpahii ftssivgirg vpnvavysat kgaidsfars larefgpkni 181hvncvnpgtt rtemtkgvdl aafgdvpikg wievdaiada vlflikskni tgqslvvdng 241fgvSEQ ID NO 3: Candida vaccinii CBS7318 protein sequence carbonyl reductase1 mrstpnalvt ggsrgigaaa aiklaeagys vtlasrgldk lnevkaklpv vkqgqehhvw 61qldlsdvqaa lefkgaplpa skydlfvsna gvatfsptae hddkdwqnii avnltspiai 121tkalvkavge rsndnpfqia flssaaalrg vpqtavysat kagldgftrs lakelgpkgi 181hvnivhpgwt qtemtagvde prdtpipgwi qpeaiaeaiv ylaksknitg tnivvdnglt 241 iSEQ ID NO 4: Candida magnoliae CBS6396 protein sequence carbonyl reductase1 mnalvtggsr gigeaiatkl aedgysvtia srgidqlnkv kaklpvvreg qthhvwqldl 61sdaeaassfk gaplpassyd vlvnnagvtd pspiakqsds eihklfsvnl lspvaltkty 121vqavtgkpre tpahiifiss gvairgypnv avysatksgl dgfmrslare lgpegvhvnt 181vspgltktem asgvslddfp pspiggwigp eaiadavryl vksknitgti lsvdngitv

NUCLEIC ACID SEQUENCESSEQ ID NO 5: Oryctolagus cuniculus from rabbit DSMZ 22167 1atggcttcat ctggcgtaac acgccgtgat ccgctggcca acaaagtcgc tattgtcact 61gcgtcgaccg atggcatcgg actggcgatt gcgcgtcgcc ttgctcagga cggggctcac 121gtggtaatct cttcgcgtaa acagcaaaat gtagatcgtg ccgttgctgc cctgcaagca 181gaaggtctgt ccgtaactgg tactgtgtgc catgtcggga aagccgagga ccgtgaacgt 241ctggttgcga cggcccttaa tcttcatggc ggtatcgata tcctggtgag taacgcggcc 301gtcaatccgt ttttcggtaa gttaatggac gtcaccgaag aggtgtggga taaaattctg 361gacatcaacg tgaaagcaat ggcgttgatg accaaagcgg tggttccaga aatggaaaaa 421cgcggtgggg gctcagttgt cattgtggcc agcattgcag cctttaatcc atttagcggc 481ttaggtccgt acaatgtgag taaaacggca ttggttggcc tgaccaagaa cctggcattg 541gagttagcag cgcagaacat tcgtgttaac tgtttagcgc cgggcctgat taagacatca 601ttcagtaagg cactgtggga ggataaagct caggaggaaa atatcattca gaaactgcgt 661attcgccgtc tgggaaaacc ggaagaatgt gcaggtatcg ttagctttct gtgctctgaa 721gatgcgtcct atattacggg tgaaaccgta gtggttgccg gcggagcgcc gagccgcctgSEQ ID NO 6: Candida magnoliae DSMZ 22052 nucleic acid sequence carbonyl reductase1 atgtctgcta cttcgaacgc tcttatcact ggtgccagcc gcggaatggg cgaggccaca 61gctattaagc ttgcccttga ggggtacagc gtcacccttg catcacgcgg tattgagcag 121ctcaatgcca tcaaggaaaa actacccatc gtgaagaagg gccagcagca ctacgtttgg 181cagctcgatc ttagtgacat cgaggcggct tccaccttca agggggctcc tctgcctgcc 241agcagctacg acgtgttctt cagcaacgcc ggtgtggtgg actttgctcc gttcgcagac 301caaagcgaga ctgcgcaaaa ggacctgttc acggttaacc tgctgtcgcc tgttgcgttg 361accaagacca ttgttaaggc catcgccgac aagccccgcg agacgcctgc tcacattatc 421ttcacctcgt ccattgtcgg aattcgcggt gttcccaacg tggcggtcta cagcgccacc 481aagggcgcga ttgacagctt tgcgcgctcg cttgctcgtg agttcggtcc caagaacatc 541cacgttaact gcgtgaaccc gggcacgacg cgcaccgaga tgacaaaggg cgttgatctc 501gcggctttcg gcgatgttcc tatcaagggc tggatcgagg tcgatgcgat tgccgacgct 661gtgctctttt tgatcaagtc caagaacatc actggccagt cgctcgttgt tgacaacgga 721ttcggtgttt aaSEQ ID NO 7: Candida vaccinii CBS7318 nucleic acid sequence carbonyl reductase1 atgaggtcga cacctaacgc ccttgtgact ggcggcagcc gcggcattgg cgcggccgct 61gcaattaaac tcgccgaggc aggctacagc gtgacgctcg cgtcgcgcgg tctcgacaag 121ctcaacgagg tgaaggccaa gcttcctgtc gtgaagcagg gccaggagca ccatgtatgg 181cagcttgatc tcagcgacgt gcaggccgcg ctcgagttca agggcgcacc gctgcccgcg 241agtaagtacg atttgtttgt ctcgaacgcc ggcgtggcta ctttctcgcc aacggctgag 301catgacgaca aggactggca gaacattatt gccgtgaact tgacatcgcc cattgccatt 361acgaaggcgc tcgttaaggc cgttggcgag cgctcaaacg ataacccgtt tcagatcgcg 421ttcctgtcat cggcggccgc cctgcgcggt gtgccgcaga ccgctgttta cagcgctacg 481aaggccggcc tcgacggctt cacgcgctcg ctcgccaagg agctcggccc aaagggcatc 541catgtgaaca tcgtacaccc tggatggacg cagaccgaga tgactgcggg tgtagatgag 601cctagggata cgcccatccc gggctggatc cagccggaag ccatcgccga ggccattgtg 661tatctcgcga agtcaaagaa catcacggga acgaacatcg ttgtcgacaa cggcctgact 721atttaaSEQ ID NO 8: Candida magnoliae CBS6396 nucleic acid sequence carbonyl reductase1 atgaacgctc tagtgaccgg tggtagccgt ggcattggcg aggcgatcgc gaccaagctg 61gccgaagatg gctacagcgt gacaatcgcc tcgcgcggaa tcgatcagct caacaaggta 121aaggctaaac ttccggttgt gagggagggc cagacccacc acgtgtggca gcttgatttg 181agcgacgccg aggccgcgtc gtccttcaag ggcgctcctt tgccagcaag cagctacgat 241gtccttgtca acaacgccgg agtaacggat ccgagtccca ttgcgaagca gtcggatagc 301gagattcaca agctgtttag cgtgaatctg ctgtcaccag ttgctttgac aaagacgtac 361gtccaggcgg ttaccggaaa gcctcgtgag acgccagctc acattatttt tatctcgtca 421ggcgttgcca ttcgaggcta cccaaacgtc gctgtatact cggctactaa gagcgggctc 481gacggtttca tgaggtctct ggcgcgcgag cttggccccg agggcgtcca tgtgaacact 541gtcagcccgg gtctcaccaa aaccgagatg gccagcggcg tcagcctcga cgacttcccg 601ccatcgccga ttgggggctg gatccagccc gaggccatcg ctgatgcagt gaggtacctg 661gtgaagtcga agaacatcac aggcacgatt ctgtcagttg acaacggaat cacggtttaa

What is claimed is:
 1. A method for preparing carbamic acidaryl-2-tetrazolyl ethyl ester of Chemical Formula 1, comprising:subjecting an arylketone of Chemical Formula 2, to enantioselectiveenzymatic reduction in a reaction mixture comprising said compound ofChemical Formula 2, an oxidoreductase having at least 90% homology withamino acid sequence SEQ ID NO: 4, NADH or NADPH as a cofactor that isoxidized during the reduction process and continuously regenerated, asecond substrate comprising a secondary alcohol of the Formula RR′CHOH,wherein R and R′ are independent from one another and have from 1 to 10carbon atoms, and a suitable buffer to form an alcohol compound of(R)-configuration of Chemical Formula 3; and carbamating said alcohol

wherein, R₁ and R₂ are independently selected from the group consistingof hydrogen, halogen, perfluoroalkyl, an alkyl of 1 to 8 carbon atoms, athioalkoxy of 1 to 8 carbon atoms, and an alkoxy of 1 to 8 carbon atoms;and one of A₁ and A₂ is CH with the other being N.
 2. The methodaccording to claim 1, wherein the oxidoreductase is encoded by nucleicacid sequence SEQ ID NO:
 8. 3. The method according to claim 2, whereinthe oxidoreductase can be isolated from Candida magnolia, Candidavaccinii or Oryctolagus cuniculus.
 4. The method according to claim 1,wherein the oxidoreductase is purified or in a microbial cells.
 5. Themethod according to claim 4, wherein the oxidoreductase is present inthe microbial cell and is permeabilized.
 6. The method according toclaim 5, wherein the microbial cells are transformed Escherichia colicells.
 7. The method according to claim 1, wherein the the the oxidizedcofactor is regenerated to the cofactor by the oxidation of said secondsubstrate.
 8. The method according to claim 1, wherein said secondsubstrate is a secondary alcohol selected from the group consisting of2-propanol, 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-heptanol and2-octanol.
 9. The method according to claim 1, wherein saidoxidoreductase affects both the reduction of the arylketone of ChemicalFormula 2 and the oxidation of the second substrate.
 10. The methodaccording to claim 1, wherein the carbamating step comprises reactingthe alcohol compound of (R)-configuration of Chemical Formula 3 withinorganic cyanate and an organic acid.
 11. The method according to claim1, wherein the carbamating step comprises hydrolyzing a productresulting from the reaction between the alcohol compound of(R)-configuration of Chemical Formula 3 and an isocyanate compoundselected from the group consisting of chlorosulfonic isocyanate,trichloroacetyl isocyanate and trimethylsilyl isocyanate.
 12. The methodaccording to claim 1, wherein the carbamating step comprises introducingammonia into a product resulting from the reaction between the alcoholcompound of (R)-configuration of Chemical Formula 3 and a carbonylcompound comprising 1,1′-carbonyldiimidazole, carbamoylhalide, N,N-disuceinimidyl carbonate, phosgene, triphosgene, or chloroformate. 13.The method according to claim 1, further comprising crystallizing thecompound of Formula
 1. 14. The method according to claim 13, wherein thecrystallizing step comprises: adding to a reaction product asolubilizing agent selected from the group consisting of acetone,acetonitrile, tetrahydrofuran, ethyl acetate, dichloromethane,chloroform, 1,4-dioxane, an alcohol of 1 to 4 carbon atoms and mixturesthereof; and adding a precipitating agent thereto selected from thegroup consisting of water, an alcohol of 1 to 4 carbon atoms,diethylether, pentane, hexane, cyclohexane, heptane and mixturesthereof.
 15. The method according to claim 1, further comprising: thestep of preparing the arylketone of Chemical Formula 2 by a substitutionreaction between an arylketone of Chemical Formula 4 and a tetrazole ofChemical Formula 5:

wherein, R₁ and R₂ are as defined in claim 1; and X is a leaving groupselected from among halides and sulfonates.
 16. A method according toclaim 15, further comprising a crystallizing step comprising: adding asolubilizing agent selected from the group consisting of acetone,acetonitrile, tetrahydrofuran, ethyl acetate, dichloromethane,chloroform, 1,4-dioxane, an alcohol of 1 to 4 carbon atoms and mixturesthereof to a product obtained by the substitution reaction; and adding aprecipitating agent selected from the group consisting of water analcohol of 1 to 4 carbon atoms, diethylether, pentane, hexane,cyclohexane, heptanes and mixtures thereof.
 17. A method for preparingan alcohol compound of Chemical Formula 3, through the enantioselectiveenzymatic reduction of an arylketone of Chemical Formula 2:

wherein, R₁ and R₂ are independently selected from a group consisting ofhydrogen, halogen, perfluoroalkyl, an alkyl of 1 to 8 carbon atoms, athioalkoxy of 1 to 8 carbon atoms, and an alkoxy of 1 to 8 carbon atoms,one of A₁ and A₂ is CH with the other being N; and said reduction iscarried out in a reaction mixture comprising said compound of ChemicalFormula 2, an oxidoreductase having at least 90% homology with aminoacid sequences SEQ ID NO:4, NADH or NADPH as a cofactor that is oxidizedduring the reduction process and continuously regenerated, a secondsubstrate comprising a secondary alcohol of the Formula RR′CHOH, whereinR and R′ are independent from one another and have from 1 to 10 carbonatoms, and a suitable buffer.
 18. The method according to claim 17,wherein the oxidoreductase is encoded by nucleic acid sequence SEQ IDNO:8.
 19. The method according to claim 17, wherein the oxidoreductaseis isolated from Candida magnolia, Candida vaccinii or Oryctolaguscuniculus.
 20. The method according to claim 1, wherein theoxidoreductase is partially purified.
 21. The method according to claim1, wherein the oxidoreductase is a microbial cell lysate.